The work described in this proposal represents a truly new strategy for improving the targeting of lipid-based delivery vehicles to specific sites in vivo. Previous studies on intravenous delivery have demonstrated the need to have prolonged circulation times in order for vehicles to accumulate in target tissues (e.g., tumors). The predominant strategy for achieving prolonged circulation times is via PEGylation, but studies have shown that this strategy can compromise delivery, suggesting that it would be advantageous to develop alternative strategies. Our previous work has demonstrated the ability of lipid nanoparticles containing high cholesterol contents to avoid aggregation in blood and accumulate in tumors. In addition, our most recent work has demonstrated that cholesterol can form nanodomains at very high concentrations, mimicking lipid rafts that endow biological membranes with multiple functionalities. The proposed studies exploit this lipid mixing behavior to create regions of the delivery vehicle that can be used as anchoring sites for targeting ligands. A significant advantage of this approach is that cholesterol nanodomains appear to remain free of protein, suggesting that ligands located within nanodomains will not be compromised by protein fouling upon intravenous administration. Our preliminary results are consistent with this hypothesis, and demonstrate that ligands located within the nanodomain dramatically improve transfection rates in cell culture and in vivo, in contrast to the identical ligand when it is excluded from the nanodomain. The experiments outlined in this proposal investigate the effects of lipid composition on nanodomain formation in order to fully exploit lipid mixing behavior to improve targeting. The proposed studies will also optimize ligand density, spacer length, uptake and delivery in cell culture, before testing this strategy in a xenograft mouse model. Furthermore, the experiments will assess the ability of two small molecule ligands (KYT-0353, anisamide) to deliver three different drug cargoes (plasmid, siRNA, and doxorubicin) both in vitro and in vivo. Accordingly, the proposed studies will thoroughly investigate the use of nanodomains to improve vehicle targeting, and assess the applicability of this approach to different drug types.